2.8 Focus reduction neutralization assay (FRNT)
The cross-neutralizing activity against G1 and G2 PEDV strains between the anti-G1-NTD and –G2-NTD serum was evaluated using a focus reduction neutralization test (FRNT) with minor modifications (Vaigya et al., 2010). Initially, 500 μl of infection medium-diluted virus stock solution (200 pfu/ml) was mixed with equal volume of twofold serial infection medium-diluted either the polyclonal antisera or the control syngeneic sera, and then incubated for 1 h at 37ºC. The mixture was added to confluent Vero cells in 6-well plates, which were initially washed twice with infection medium, then incubated for 2 h at room temperature to allow adsorption of the virus. After incubation, the medium was removed. The infected cells were overlaid with 1% sodium carboxymethyl cellulose that was dissolved infection medium and incubated for 36 h. The overlaid medium was removed and the cells were fixed as in IFA, and incubated with anti-G1-NTD or –G2-NTD serum for 1 h at 37 ºC. After being washed three times with PBS, the monolayer cells were incubated with HRP-conjugated goat anti-mouse IgG (1:1000) for 1 h at 37 ºC. Finally, virus-specific foci were positively stained with 3, 3 - diaminobenzidine (DAB) and counted under microscope. Serum neutralization titers were expressed as the reciprocals of the highest serum dilution resulting in reduction of 50% focus forming unit.